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• W2146754019 abstract "Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys158-Ile159 (P1-P1′) peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (kcat/Km 4.8 × 105 m–1 s–1) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 ± 1.9 nm uPA h–1, which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression. Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys158-Ile159 (P1-P1′) peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (kcat/Km 4.8 × 105 m–1 s–1) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 ± 1.9 nm uPA h–1, which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression. Hepsin is a type II transmembrane serine protease expressed on the surface of epithelial cells. The 417-amino acid protein is composed of a short N-terminal cytoplasmic domain, a transmembrane domain, and a single scavenger receptor cysteine-rich domain that packs tightly against the C-terminal protease domain (1Somoza J.R. Ho J.D. Luong C. Ghate M. Sprengeler P.A. Mortara K. Shrader W.D. Sperandio D. Chan H. McGrath M.E. Katz B.A. Structure. 2003; 11: 1123-1131Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The physiologic function of hepsin remains unknown. Despite its expression in the very early stages of embryogenesis (2Vu T.K. Liu R.W. Haaksma C.J. Tomasek J.J. Howard E.W. J. Biol. Chem. 1997; 272: 31315-31320Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), hepsin-deficient mice were viable and developed normally (3Yu I.S. Chen H.J. Lee Y.S. Huang P.H. Lin S.R. Tsai T.W. Lin S.W. Thromb. Haemostasis. 2000; 84: 865-870Crossref PubMed Scopus (38) Google Scholar, 4Wu Q. Yu D. Post J. Halks-Miller M. Sadler J.E. Morser J. J. Clin. Investig. 1998; 101: 321-326Crossref PubMed Scopus (77) Google Scholar). The studies further showed that hepsin was not essential for liver regeneration and for coagulation-related physiological functions (3Yu I.S. Chen H.J. Lee Y.S. Huang P.H. Lin S.R. Tsai T.W. Lin S.W. Thromb. Haemostasis. 2000; 84: 865-870Crossref PubMed Scopus (38) Google Scholar, 4Wu Q. Yu D. Post J. Halks-Miller M. Sadler J.E. Morser J. J. Clin. Investig. 1998; 101: 321-326Crossref PubMed Scopus (77) Google Scholar). However, hepsin has been implicated in ovarian cancer (5Tanimoto H. Yan Y. Clarke J. Korourian S. Shigemasa K. Parmley T.H. Parham G.P. O'Brien T.J. Cancer Res. 1997; 57: 2884-2887PubMed Google Scholar) and prostate cancer (6Dhanasekaran S.M. Barrette T.R. Ghosh D. Shah R. Varambally S. Kurachi K. Pienta K.J. Rubin M.A. Chinnaiyan A.M. Nature. 2001; 412: 822-826Crossref PubMed Scopus (1430) Google Scholar, 7Luo J. Duggan D.J. Chen Y. Sauvageot J. Ewing C.M. Bittner M.L. Trent J.M. Isaacs W.B. Cancer Res. 2001; 61: 4683-4688PubMed Google Scholar, 8Magee J.A. Araki T. Patil S. Ehrig T. True L. Humphrey P.A. Catalona W.J. Watson M.A. Milbrandt J. Cancer Res. 2001; 61: 5692-5696PubMed Google Scholar, 9Stamey T.A. Warrington J.A. Caldwell M.C. Chen Z. Fan Z. Mahadevappa M. McNeal J.E. Nolley R. Zhang Z. J. Urol. 2001; 166: 2171-2177Crossref PubMed Scopus (166) Google Scholar, 10Stephan C. Yousef G.M. Scorilas A. Jung K. Jung M. Kristiansen G. Hauptmann S. Kishi T. Nakamura T. Loening S.A. Diamandis E.P. J. Urol. 2004; 171: 187-191Crossref PubMed Scopus (105) Google Scholar, 11Welsh J.B. Sapinoso L.M. Su A.I. Kern S.G. Wang-Rodriguez J. Moskaluk C.A. Frierson Jr., H.F. Hampton G.M. Cancer Res. 2001; 61: 5974-5978PubMed Google Scholar), where several gene expression studies have identified it as one of the most highly induced genes. Hepsin RNA levels were found to be low in normal prostate and benign hyperplasia but strongly increased in prostate carcinoma, particularly in advanced stages (8Magee J.A. Araki T. Patil S. Ehrig T. True L. Humphrey P.A. Catalona W.J. Watson M.A. Milbrandt J. Cancer Res. 2001; 61: 5692-5696PubMed Google Scholar, 9Stamey T.A. Warrington J.A. Caldwell M.C. Chen Z. Fan Z. Mahadevappa M. McNeal J.E. Nolley R. Zhang Z. J. Urol. 2001; 166: 2171-2177Crossref PubMed Scopus (166) Google Scholar, 10Stephan C. Yousef G.M. Scorilas A. Jung K. Jung M. Kristiansen G. Hauptmann S. Kishi T. Nakamura T. Loening S.A. Diamandis E.P. J. Urol. 2004; 171: 187-191Crossref PubMed Scopus (105) Google Scholar). Hepsin protein staining with a monoclonal anti-hepsin antibody showed that hepsin expression was highest at sites of bone metastasis and in late stage primary tumors (12Xuan J.A. Schneider D. Toy P. Lin R. Newton A. Zhu Y. Finster S. Vogel D. Mintzer B. Dinter H. Light D. Parry R. Polokoff M. Whitlow M. Wu Q. Parry G. Cancer Res. 2006; 66: 3611-3619Crossref PubMed Scopus (69) Google Scholar), which is consistent with the finding that increased hepsin RNA levels correlated with higher Gleason grades and tumor progression (7Luo J. Duggan D.J. Chen Y. Sauvageot J. Ewing C.M. Bittner M.L. Trent J.M. Isaacs W.B. Cancer Res. 2001; 61: 4683-4688PubMed Google Scholar, 8Magee J.A. Araki T. Patil S. Ehrig T. True L. Humphrey P.A. Catalona W.J. Watson M.A. Milbrandt J. Cancer Res. 2001; 61: 5692-5696PubMed Google Scholar, 9Stamey T.A. Warrington J.A. Caldwell M.C. Chen Z. Fan Z. Mahadevappa M. McNeal J.E. Nolley R. Zhang Z. J. Urol. 2001; 166: 2171-2177Crossref PubMed Scopus (166) Google Scholar, 10Stephan C. Yousef G.M. Scorilas A. Jung K. Jung M. Kristiansen G. Hauptmann S. Kishi T. Nakamura T. Loening S.A. Diamandis E.P. J. Urol. 2004; 171: 187-191Crossref PubMed Scopus (105) Google Scholar, 13Chen Z. Fan Z. McNeal J.E. Nolley R. Caldwell M.C. Mahadevappa M. Zhang Z. Warrington J.A. Stamey T.A. J. Urol. 2003; 169: 1316-1319Crossref PubMed Scopus (75) Google Scholar). In contrast, using a different antibody Dhanasekaran et al. (6Dhanasekaran S.M. Barrette T.R. Ghosh D. Shah R. Varambally S. Kurachi K. Pienta K.J. Rubin M.A. Chinnaiyan A.M. Nature. 2001; 412: 822-826Crossref PubMed Scopus (1430) Google Scholar) found the strongest hepsin expression in high grade prostate intraneoplastic lesions and lower expression in primary carcinoma and in metastatic lesions. These studies raised the question of whether hepsin is involved in prostate cancer. In vitro studies did not provide clear answers, and depending on the experimental conditions used, hepsin was found to promote, inhibit, or not affect tumor cell growth (12Xuan J.A. Schneider D. Toy P. Lin R. Newton A. Zhu Y. Finster S. Vogel D. Mintzer B. Dinter H. Light D. Parry R. Polokoff M. Whitlow M. Wu Q. Parry G. Cancer Res. 2006; 66: 3611-3619Crossref PubMed Scopus (69) Google Scholar, 14Srikantan V. Valladares M. Rhim J.S. Moul J.W. Srivastava S. Cancer Res. 2002; 62: 6812-6816PubMed Google Scholar, 15Torres-Rosado A. O'Shea K.S. Tsuji A. Chou S.H. Kurachi K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7181-7185Crossref PubMed Scopus (90) Google Scholar). Evidence for a role of hepsin in prostate cancer came from a recent study by Klezovitch et al. (16Klezovitch O. Chevillet J. Mirosevich J. Roberts R.L. Matusik R.J. Vasioukhin V. Cancer Cell. 2004; 6: 185-195Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) demonstrating that in a mouse model of non-metastasizing prostate cancer, overexpression of hepsin led to primary tumor progression and metastasis. Intriguingly, hepsin overexpression was associated with basement membrane disruption (16Klezovitch O. Chevillet J. Mirosevich J. Roberts R.L. Matusik R.J. Vasioukhin V. Cancer Cell. 2004; 6: 185-195Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) pointing toward the possibility that hepsin activity is somehow linked to the degradation of basement membrane components. In vitro, hepsin was able to convert the latent growth factor pro-hepatocyte growth factor (pro-HGF) 2The abbreviations used are: HGF, hepatocyte growth factor; HGFA, HGF activator; KD1, Kunitz domain-1 derived from HAI-1B; pNA, para-nitroanilide; uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; pro-uPA, zymogen form of uPA; pro-tPA, zymogen form of tPA; pro-HGF, single chain form of HGF; TAMRA, 6-carboxytetramethylrhodamine; FAM, 6-carboxyfluorescein; PBS, phosphate-buffered saline. into its active two-chain form (HGF), which induced Met receptor signaling (17Herter S. Piper D.E. Aaron W. Gabriele T. Cutler G. Cao P. Bhatt A.S. Choe Y. Craik C.S. Walker N. Meininger D. Hoey T. Austin R.J. Biochem. J. 2005; 390: 125-136Crossref PubMed Scopus (147) Google Scholar, 18Kirchhofer D. Peek M. Lipari M.T. Billeci K. Fan B. Moran P. FEBS Lett. 2005; 579: 1945-1950Crossref PubMed Scopus (131) Google Scholar). Because the HGF/Met pathway has been implicated in invasive tumor growth and metastasis, it is possible that overexpression of hepsin activates the HGF/Met axis in prostate cancer. Hepsin was also shown to cleave other substrates in vitro, mainly coagulation-related proteins (17Herter S. Piper D.E. Aaron W. Gabriele T. Cutler G. Cao P. Bhatt A.S. Choe Y. Craik C.S. Walker N. Meininger D. Hoey T. Austin R.J. Biochem. J. 2005; 390: 125-136Crossref PubMed Scopus (147) Google Scholar, 19Kazama Y. Hamamoto T. Foster D.C. Kisiel W. J. Biol. Chem. 1995; 270: 66-72Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). However, their role in tumorigenesis is not known. In view of the basement membrane defects that were associated with hepsin overexpression in the mouse prostate, we hypothesized that hepsin might activate protease zymogens that are directly linked to basement membrane degradation. It was known from previous studies that hepsin does not activate plasminogen (17Herter S. Piper D.E. Aaron W. Gabriele T. Cutler G. Cao P. Bhatt A.S. Choe Y. Craik C.S. Walker N. Meininger D. Hoey T. Austin R.J. Biochem. J. 2005; 390: 125-136Crossref PubMed Scopus (147) Google Scholar, 18Kirchhofer D. Peek M. Lipari M.T. Billeci K. Fan B. Moran P. FEBS Lett. 2005; 579: 1945-1950Crossref PubMed Scopus (131) Google Scholar), and therefore, we considered the upstream activators of plasminogen, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) as possible candidates. The results of this study show that soluble hepsin is a potent activator of pro-uPA with a catalytic efficiency comparable with that of plasmin. Pro-uPA conversion was also measured by hepsin-expressing LnCaP prostate cancer cells, demonstrating that cellular full-length hepsin recapitulates the function of soluble hepsin. The implications of these findings with respect to the function of hepsin in prostate cancer progression are discussed. Reagents—Lys-plasmin and Lys-plasminogen were from Heamatologic Technologies Inc. (Essex Junction, VT). Pro-tPA was from Biodesign International (Saco, ME), uPA (high molecular weight form) was from American Diagnostica (Greenwich, CT), pro-uPA was from Cortex Biochem (San Leandro, CA), and PAI-1 was from Molecular Innovations (Southfield, MI). The chromogenic substrates S2444, S2765, and S2366 were from Diapharma (Westchester, OH). T.in.Pro cells were from Expression System LLC (Woodland, CA). Nickel-nitrilotriacetic acid resin was from Qiagen Inc. (Chatsworth, CA), and Q-Sepharose and benzamidine-Sepharose 4 Fast Flow was from GE Healthcare. Construction, Expression, and Purification of Recombinant Proteins—A soluble form of hepsin comprising the entire extracellular domain was produced by use of a baculovirus expression system. A secreted His-tagged hepsin cDNA was constructed by fusion of the cDNA coding for the signal sequence of honeybee melittin (Met1-Tyr20) with the cDNA coding for the extracellular domain of human hepsin (Arg45-Leu417). The final cDNA construct was inserted in a baculovirus expression vector under the control of a polyhedrin promoter and expressed in T.in.Pro cells. Hepsin was purified by nickel-nitrilotriacetic acid affinity chromatography essentially as described (20Kirchhofer D. Peek M. Li W. Stamos J. Eigenbrot C. Kadkhodayan S. Elliott J.M. Corpuz R.T. Lazarus R.A. Moran P. J. Biol. Chem. 2003; 278: 36341-36349Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Hepsin-containing medium was conditioned with 1 mm sodium azide, 0.3 m NaCl, and 15 mm imidazole and was adjusted to pH 6.5 using NaOH. Precharged nickel-nitrilotriacetic acid resin was added to media (4 ml resin/liter medium). Batch absorption was performed by gently stirring at 4 °C for 2 h. After allowing the resin to settle for 1 h, the supernatant was decanted, and the resin was packed into a column. The column was washed with a minimum of 10 column volumes of PBS, 0.3 m NaCl, pH 7.4, then followed by 10 column volumes of 25 mm imidazole, 0.3 m NaCl, 1 mm sodium azide, pH 8.0. Proteins were eluted with 250 mm imidazole, 0.3 m NaCl, 1 mm sodium azide, pH 8.0. Pooled fractions were purified further using either ion-exchange chromatography on a Q-Sepharose FF ion or affinity chromatography on a benzamidine-Sepharose 4 Fast Flow column. The matriptase protease domain was expressed in Escherichia coli and purified as described (20Kirchhofer D. Peek M. Li W. Stamos J. Eigenbrot C. Kadkhodayan S. Elliott J.M. Corpuz R.T. Lazarus R.A. Moran P. J. Biol. Chem. 2003; 278: 36341-36349Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). HGFA and soluble HAI-2 were recombinantly expressed and purified as described (18Kirchhofer D. Peek M. Lipari M.T. Billeci K. Fan B. Moran P. FEBS Lett. 2005; 579: 1945-1950Crossref PubMed Scopus (131) Google Scholar, 20Kirchhofer D. Peek M. Li W. Stamos J. Eigenbrot C. Kadkhodayan S. Elliott J.M. Corpuz R.T. Lazarus R.A. Moran P. J. Biol. Chem. 2003; 278: 36341-36349Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The active site concentration of hepsin, matriptase, and HGFA was determined by using the potent Kunitz domain inhibitor KD1 derived from HAI-1B, which was produced in E. coli as described (21Shia S. Stamos J. Kirchhofer D. Fan B. Wu J. Corpuz R.T. Santell L. Lazarus R.A. Eigenbrot C. J. Mol. Biol. 2005; 346: 1335-1349Crossref PubMed Scopus (58) Google Scholar). The active site concentrations were used for all enzymatic assays. For plasmin, the concentration provided by the supplier (Hematologic Laboratories, Inc.) was used. Monoclonal Anti-hepsin Antibody—Five Balb/c mice (Charles River Laboratories) were hyperimmunized with recombinant soluble hepsin in RIBI adjuvant (Ribi Immunochem Research Inc.). B cells from lymph nodes from five mice were fused with mouse myeloma cells (X63.Ag8.653; American Type Culture Collection, Manassas, VA) as previously described (22Hongo J.A. Mora-Worms M. Lucas C. Fendly B.M. Hybridoma. 1995; 14: 253-260Crossref PubMed Scopus (21) Google Scholar). After 10–14 days the supernatants were harvested and screened for antibody production with a hepsin binding enzyme-linked immunosorbent assay. The clone 3H10 showed high immunobinding and specificity after the second round of single cell per well cloning (Elite 1 Sorter, Beckman Coulter) and was scaled up for purification in INTEGRA CELLine 1000 (Integra Biosciences). The supernatant was purified by protein A affinity chromatography, sterile-filtered, and stored at 4 °C in PBS. Isotyping with the mono-AB-ID SP Kit (Zymed Laboratories Inc.) showed that 3H10 is an IgG1κ. Production of Hepsin Overexpressing LnCaP Cells—The human prostate carcinoma cell line, LnCaP-FGC (LnCaP), was obtained from American Type Culture Collection. The cells were cultured in RPMI 1640 medium (American Type Culture Collection) plus 10% fetal bovine serum (Sigma-Aldrich). A LnCaP clone that stably expressed the firefly luciferase gene (LnCaP-luc) was used for hepsin transfection experiments. To establish the LnCaP-luc cell line, the luciferase gene was subcloned as an EcoRI/XhoI cDNA fragment inserted into the pMSCVneo expression vector (BD Biosciences Clontech, Mountain View, CA). LnCaP cells were transfected with the luciferase construct using Lipofectamine 2000 (Invitrogen). The cells were selected with 500 μg/ml Geneticin (Invitrogen), and clones were screened for bioluminescence activity by using the Luclite kit (PerkinElmer Life Sciences). The clone LnCaP-luc, which produced the strongest luminescence signal, was chosen for hepsin transfection experiments. The cDNA of full-length hepsin was inserted into a mammalian expression vector containing the puromycin resistance gene for antibiotic selection (Genentech, South San Francisco, CA). The LnCaP-luc clone was transfected with the construct encoding full-length hepsin with a C-terminal FLAG tag, and the cells were selected with 0.5 μg/ml puromycin (Sigma-Aldrich). The clones were analyzed by fluorescence-activated cell sorter for hepsin surface expression using an anti-FLAG monoclonal antibody (Sigma-Adrich). Two clones, the high hepsin expressor LnCaP-34 and the low hepsin expressor LnCaP-17, were selected for further experiments. To measure total hepsin expression (endogenous and transfected) on the cell surface, LnCaP-34 and LnCaP-17 cell suspensions in PBS/1% (v/v) fetal bovine serum were incubated with 10 μg/ml of 3H10 antibody or without antibody (control) for 40 min on ice. The cells were washed twice with PBS before incubation with R-phycoerythrin-conjugated F(ab′)2 goat anti-mouse IgG (Jackson Immunoresearch Laboratories Inc.) diluted 1:1000 in PBS, 1% fetal bovine serum (v/v). After 30 min on ice the cells were washed with PBS, and cell pellets were resuspended in 1% formalin (Richard Allen Scientific). Antibody binding was measured on a FACSscan (BD Biosciences). Real-time Reverse Transcription-PCR—Total RNA was isolated from LnCaP-17 and LnCaP-34 cells using RNeasy Mini Kit (Qiagen, Valencia, CA). Gene expression analysis was performed by real-time reverse transcription PCR (TaqMan) on a model 7500 sequence detector (ABI-PerkinElmer, Foster City, CA). To specifically measure endogenous hepsin we used primers recognizing sequences in the 3′-untranslated region of the hepsin gene. To measure total hepsin (endogenous plus transfected hepsin) we used primers recognizing sequences in the open reading frame that is common to both hepsins. The sequences of primers and probes were as follows: endogenous hepsin, forward (5′-CCCTCCAGGGTCCTCTCT-3′), reverse (5′-AGTCCCAGACAGCAGAACAATA-3′), probe (5′-(FAM)-CAGCCCCGAGACCACCCAAC-(TAMRA)-3′); total hepsin, forward (5′-GCTGTGTGGCATTGTGAGT-3′), reverse (5′-TGAGTCTTTATGGCCTGGAA-3′), probe (5′-(FAM)-AAGCCAGGCGTCTACACCAAAGTCAG-(TAMRA)-3′); matriptase, forward (5′-CTTCGGAGCCTCCTCAGT-3′), reverse (5′-GTCTCAGACCCGTCTGTTTTC-3′), probe (5′-(FAM)-CCTCCGAGCCTGGGCTTCCT-(TAMRA)-3′); glyceraldehyde-3-phosphate dehydrogenase, forward (5′-GAAGGTGAAGGTCGGAGTC-3′), reverse (5′-GAAGATGGTGATGGGATTTC-3′), probe (5′-(FAM)-CAAGCTTCCCGTTCTCAGCC-(TAMRA)-3′). The reverse transcription was carried out at 48 °C for 30 min followed by heat activation of AmpliTaq Gold at 95 °C for 10 min. The thermal cycling proceeded with 40 cycles of 95 °C for 0.5 min and 60 °C for 1 min. All samples were run in duplicate. The results were quantified using the standard curve method according to the manufacturer's instruction (ABI-PerkinElmer). All gene expression levels were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. Enzymatic Assays with HAI-2 and the Small Molecule Inhibitor HI-10331—The reversible active site inhibitor HI-10331 (a gift from Alan Olivero, Genentech, Inc.) or HAI-2 was incubated with 0.5 nm hepsin, 0.5 nm matriptase, and 10 nm uPA for 30 min in Hepes buffer (20 mm Hepes, pH 7.5, 150 mm NaCl, 5 mm CaCl2, 0.01% Triton X-100) at room temperature. The chromogenic substrates S2366 (for hepsin), S2765 (for matriptase), and S2444 (for uPA) were added at a concentration corresponding to their respective Km values, which were determined in separate experiments. After substrate addition, the linear rates of the increase in absorbance at 405 nm were measured on a kinetic microplate reader (Molecular Devices, Sunnyvale, CA). For HI-10331, the inhibitor concentration that gave a 50% inhibition of the enzymatic activity (IC50) was determined by fitting the data to a four-parameter equation (Kaleidagraph Version 3.6, Synergy Software, Reading, PA). The Ki values were calculated according to the relationship Ki = IC50/(1 + [S]/Km) (23Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12330) Google Scholar) using the experimentally determined IC50 and Km values for each enzyme-substrate pair. For HAI-2, the apparent Ki values (Ki*) were determined by fitting the data to the equation for tight binding inhibition (24Morrison J.F. Biochim. Biophys. Acta. 1969; 185: 269-286Crossref PubMed Scopus (736) Google Scholar, 25Olivero A.G. Eigenbrot C. Goldsmith R. Robarge K. Artis D.R. Flygare J. Rawson T. Sutherlin D.P. Kadkhodayan S. Beresini M. Elliott L.O. DeGuzman G.G. Banner D.W. Ultsch M. Marzec U. Hanson S.R. Refino C. Bunting S. Kirchhofer D. J. Biol. Chem. 2005; 280: 9160-9169Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), ViVo=1-([E]+[I]+Ki*)-([E]+[I]+Ki*)2-(4[E][I])2[E] where vi/vo is the fractional activity, [E] is the enzyme concentration, and [I] is the inhibitor concentration. Pro-uPA Activation by LnCaP Cells—Confluent LnCaP-34 and LnCaP-17 cells were washed with HBSA-glucose buffer (20 mm Hepes pH 7.5, 150 mm NaCl, 5 mm CaCl2, 0.05 mg/ml BSA, 5 mm glucose), and 0.8 ml of 100 nm pro-uPA in prewarmed HBSA-glucose buffer was added to the cell layers. The inhibitors KD1 or HI-10331 were added to give final concentrations of 1 and 10 μm, respectively. The culture plates were kept at 37 °C, and 50-μl samples were withdrawn at different time points and supplemented with 0.2 ml of 0.625 mm S2444 in Hepes buffer, and the increase in absorbance at 405 nm was measured on a kinetic microplate reader. Cell numbers were determined at the end of the experiments. The concentration of formed uPA in each sample was calculated from a standard curve of enzymatically converted pro-uPA and normalized to 106 cells. After subtracting the background levels of uPA formed in the absence of cell layer, the linear rates of uPA formation/106 cells were determined. The pro-uPA activation was strictly dependent on the presence of the cells, since the samples taken at different time points did not convert any additional pro-uPA. Also, the enzymatic activity toward S2444 of the withdrawn samples was entirely due to uPA activity and not to hepsin or other proteases released from the cell surface, since the chromogenic activity of the samples was not inhibited by the addition of the hepsin inhibitor KD1 but was completely inhibited by the uPA inhibitor PAI-1 (data not shown). For immunoblotting experiments, confluent LnCaP-34 cell layers were washed as above and incubated in HBSA-glucose buffer with 30 nm pro-uPA at 37 °C in the presence of 1 μm KD1 or without. After 1, 3, and 5 h, aliquots were taken and immediately added to SDS sample buffer. The proteins were separated by SDS-PAGE and transferred onto nitrocellulose filters using the Bio-Rad Semi-Dry Transfer system. Pro-uPA and uPA were visualized by using a rabbit polyclonal anti-uPA antibody (Cell Sciences, Canton, MA) followed by horseradish peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch Laboratory, West Grove, PA) and ECL (GE Healthcare) enhancement. Analysis of Pro-uPA and Pro-tPA Activation by SDS-PAGE—Pro-uPA at a concentration of 1.5 μm was incubated with 15 nm hepsin or 15 nm plasmin in Hepes buffer at room temperature. After 4 and 60 min, aliquots of the reaction mixture were taken and added to 6× SDS sample buffer. The samples were analyzed by SDS-PAGE using a 4–20% gradient gel (Invitrogen). Protein was visualized after staining with SimplyBlue Safe Stain (Invitrogen). Experiments with pro-tPA (1.5 μm) were carried out identically, except that the buffer used was 50 mm sodium phosphate, pH 7.5, 200 mm arginine, 0.01% Tween 20. Determination of First-order Rate Constants—The Km value for pro-uPA activation by plasmin is in the micromolar range (26Collen D. Zamarron C. Lijnen H.R. Hoylaerts M. J. Biol. Chem. 1986; 261: 1259-1266Abstract Full Text PDF PubMed Google Scholar, 27Lijnen H.R. Van Hoef B. Collen D. Eur. J. Biochem. 1987; 169: 359-364Crossref PubMed Scopus (48) Google Scholar, 28Wolf B.B. Vasudevan J. Henkin J. Gonias S.L. J. Biol. Chem. 1993; 268: 16327-16331Abstract Full Text PDF PubMed Google Scholar). This is consistent with our findings in an attempt to determine Km values for plasmin, hepsin, and matriptase. However, because the pro-uPA stock solution from the supplier (0.8 mg/ml) was not sufficiently high for accurate Km measurements and the calculation of the catalytic efficiencies, we chose to determine the first-order rate constant k as a measure of pro-uPA conversion efficiency. It was ensured that the pro-uPA concentration used (30 nm) was in the range of first-order kinetics for the enzymes tested, i.e. hepsin, plasmin, and matriptase. Pro-uPA (30 nm) was added to the enzymes (3 nm) in Hepes buffer to start the reaction at room temperature. At various time points, 50-μl aliquots were removed and added to 150 μl of 667 nm HAI-2 (final concentration in 250 μl was 400 nm) in Hepes buffer to stop further pro-uPA cleavage. At the concentration used, HAI-2 specifically inhibits plasmin (29Delaria K.A. Muller D.K. Marlor C.W. Brown J.E. Das R.C. Roczniak S.O. Tamburini P.P. J. Biol. Chem. 1997; 272: 12209-12214Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), HGFA (30Kawaguchi T. Qin L. Shimomura T. Kondo J. Matsumoto K. Denda K. Kitamura N. J. Biol. Chem. 1997; 272: 27558-27564Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), hepsin, and matriptase but not the newly generated uPA (Table 1). Our results on HAI-2 inhibition of plasmin and HGFA (data not shown) were consistent with the published data (29Delaria K.A. Muller D.K. Marlor C.W. Brown J.E. Das R.C. Roczniak S.O. Tamburini P.P. J. Biol. Chem. 1997; 272: 12209-12214Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30Kawaguchi T. Qin L. Shimomura T. Kondo J. Matsumoto K. Denda K. Kitamura N. J. Biol. Chem. 1997; 272: 27558-27564Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). After the addition of 50 μl of 2.5 mm S2444, the increase in absorbance at 405 nm was measured on a kinetic microplate reader. The concentration of uPA in each aliquot was calculated from a standard curve of enzymatically converted pro-uPA. The data were expressed as the decrease in the concentration of pro-uPA (log[pro-uPA]) as function of time. The first-order rate constant k was calculated from the slope using the equation log[S] =–(k/2.3)t + log[S]0, where [S] is the concentration of pro-uPA, and [S]0 is the initial pro-uPA concentration. The catalytic efficiency, kcat/Km, was then calculated using the known enzyme concentration [E] and the relationship k = (kcat/Km) [E]. The values are the average of 5 experiments ± S.D.TABLE 1Equilibrium dissociation constants for the inter" @default.
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• W2146754019 date "2006-10-01" @default.
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• W2146754019 title "Pro-urokinase-type Plasminogen Activator Is a Substrate for Hepsin" @default.
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• W2146754019 doi "https://doi.org/10.1074/jbc.m605440200" @default.
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